Bacteriological activity in unfiltered calf sera collected for tissue culture use.

Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found were Bacillus species and streptococci and 63% of the sera coagulated Limulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain of Escherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout ( Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 × 10⁸), they became septicaemic and a large amount of endotoxin (> 14 ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (> 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 μg), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Bacterial virus contamination of fetal bovine sera

Summary Thirty-seven lots of fetal bovine sera were examined for the presence ofEscherichia coli-specific bacterial viruses, and 23 were positive. No correlation was found between the presence of bacterial virus and poor growth-promoting qualities of the sera. One bacterial virus contaminant of fetal bovine serum was isolated and examined by electron microscopy.     

Bacterial host strains that support replication of somatic coliphages

Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log₁₀ fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible. This revised version was published online in June 2006 with corrections to the Cover Date.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout (Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 x 10(8], they became septicaemic and a large amount of endotoxin ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (greater than 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 micrograms), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Comparison of serological specificity of anti-endotoxin sera directed against whole bacterial cells and core oligosaccharide of Escherichia coli J5-tetanus toxoid conjugate

The rough mutants of Gram-negative bacteria are widely used to induce protective antisera but the nature of the target epitope for such antibodies is not precisely defined. Endotoxin is one of several antigens present on the surface of bacterial cells, which are able to elicit specific antibodies. We studied the specificity of antibodies produced against a conjugate of E. coli J5 endotoxin core oligosaccharide with tetanus toxoid. The use of chemically defined antigen for immunisation excludes the possibility of production of antibodies against other cell surface antigens. A comparison of this monospecific anti-endotoxin serum with antiserum against E. coli J5 whole cells was performed in order to distinguish the role that endotoxin core oligosaccharide plays in the interaction with humoral host defences from that of other potentially important Gram-negative bacterial surface antigens. The reactivity of both sera with smooth and rough lipopolysaccharides was determined in ELISA, immunoblotting and by flow cytometry. Both antisera reacted with similar specificity with most lipopolysaccharides of identical or related core type. Less distinct reactions with endotoxins of the antibacterial serum in comparison with the anti-conjugate serum were found in all serological tests. LPS of E. coli O100 that showed the strongest reactions with both sera was used to stimulate IL-6, TNFalpha and nitric oxide production by the J-774A.1 cell line. Both sera were used to inhibit that stimulation and no inhibitory effects of the examined sera in comparison with non-immune serum were observed.     

Pre‐adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates

Recent years have seen renewed interest in phage therapy – the use of viruses to specifically kill disease‐causing bacteria – because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre‐adapted all phage strains against all bacterial strains and then compared the efficacy of pre‐adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre‐adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.     

Chicken liver is a potential reservoir of bacteriophages and phage-derived particles containing antibiotic resistance genes

Poultry meat production is one of the most important agri-food industries in the world. The selective pressure exerted by widespread prophylactic or therapeutic use of antibiotics in intensive chicken farming favours the development of drug resistance in bacterial populations. Chicken liver, closely connected with the intestinal tract, has been directly involved in food-borne infections and found to be contaminated with pathogenic bacteria, including Campylobacter and Salmonella. In this study, 74 chicken livers, divided into sterile and non-sterile groups, were analysed, not only for microbial indicators but also for the presence of phages and phage particles containing antibiotic resistance genes (ARGs). Both bacteria and phages were detected in liver tissues, including those dissected under sterile conditions. The phages were able to infect Escherichia coli and showed a Siphovirus morphology. The chicken livers contained from 10³ to 10⁶ phage particles per g, which carried a range of ARGs (bla_TEM, bla_CTx-M-1, sul1, qnrA, armA and tetW) detected by qPCR. The presence of phages in chicken liver, mostly infecting E. coli, was confirmed by metagenomic analysis, although this technique was not sufficiently sensitive to identify ARGs. In addition, ARG-carrying phages were detected in chicken faeces by qPCR in a previous study of the group. Comparison of the viromes of faeces and liver showed a strong coincidence of species, which suggests that the phages found in the liver originate in faeces. These findings suggests that phages, like bacteria, can translocate from the gut to the liver, which may therefore constitute a potential reservoir of antibiotic resistance genes.     

IMPACT OF BACTERIAL MUTATION RATE ON COEVOLUTIONARY DYNAMICS BETWEEN BACTERIA AND PHAGES

Mutator bacteria are frequently found in natural populations of bacteria and although coevolution with parasitic viruses (phages) is thought to be one reason for their persistence, it remains unclear how the presence of mutators affects coevolutionary dynamics. We hypothesized that phages must themselves adapt more rapidly or go extinct, in the face of rapidly evolving mutator bacteria. We compared the coevolutionary dynamics of wild‐type Pseudomonas fluorescens SBW25 with a lytic phage to the dynamics of an isogenic mutator of P. fluorescens SBW25 together with the same phage. At the beginning of the experiment both wild‐type bacteria and mutator bacteria coevolved with phages. However, mutators rapidly evolved higher levels of sympatric resistance to phages. The phages were unable to “keep‐up” with the mutator bacteria, and these rates of coevolution declined to less than the rates of coevolution between the phages and wild‐type bacteria. By the end of the experiment, the sympatric resistance of the mutator bacteria was not significantly different to the sympatric resistance of the wild‐type bacteria. This suggests that the importance of mutators in the coevolutionary interactions with a particular phage population is likely to be short‐lived. More generally, the results demonstrate that coevolving enemies may escape from Red‐Queen dynamics.     

Phage specificity and lipopolysaccarides of stem- and root-nodulating bacteria (Azorhizobium caulinodans, Sinorhizobium spp., and Rhizobium spp.) of Sesbania spp

Phage susceptibility pattern and its correlation with lipopolysaccharide (LPS) and plasmid profiles may help in understanding the phenotypic and genotypic diversity among highly promiscuous group of rhizobia nodulating Sesbania spp.; 43 phages were from two stem-nodulating bacteria of S. rostrata and 16 phages were from root-nodulating bacteria of S. sesban, S. aegyptica and S. rostrata. Phage susceptibility pattern of 38 Sesbania nodulating bacteria was correlated with their LPS rather than plasmid profiles. Different species of bacteria (A. caulinodans- ORS571, SRS1-3 and Sinorhizobium saheli- SRR907, SRR912) showing distinct LPS subtypes were susceptible to different group of phages. Phages could also discriminate the strains of Si. saheli (SSR312, SAR610) possessing distinct LPS subtypes. Phages of Si. meliloti (SSR302) were strain-specific. All the strains of R. huautlense having incomplete LPS (insignificant O-chain) were phage-resistant. In in vitro assay, 100% of the phages were adsorbed to LPS of indicator bacterium or its closely related strain(s) only. These observations suggest the significance of LPS in phage specificity of Sesbania nodulating rhizobia. Highly specific phages may serve as biological marker for monitoring the susceptible bacterial strains in culture collections and environment.     

Establishment of phage and bacteria in a sterilized compost in a glasshouse

Summary Four strains ofRhizobium trifolii were individually inoculated to pots containing sterilized sand vermiculite mixture, half of which were seeded with red clover and half not. Pots were maintained in an ordinary glasshouse and watered with tap water.Phage was first detected after 4 months, and almost all pots contained one or more phages againstRhizobium trifolii after 9 months. The presence of plants increased the titer of phages in some pots inoculated withR. trifolii, but had no effect on the number of different phages.The pots also contained phages against soil bacteria other than Rhizobium indicating that phages are spread readily and constitute a normal part of the life cycle of soil bacteria.The number of different phages isolated from the pots was affected by the strain of Rhizobium used as inoculum.     

Lysogeny of Oenococcus oeni (syn. Leuconostoc oenos) and Study of Their Induced Bacteriophages

A large number of strains of Oenococcus oeni (formerly Leuconostoc oenos) that had been isolated from wines were checked for lysogeny with mitomycin C as inducer. As a result of this test, 45% of the strains proved to be lysogenic, suggesting that lysogeny is widespread among bacteria isolated from wines during malolactic fermentation. The sensitivity of bacteria to phages was very different, depending on the strain. All the lysogenic strains were resistant to infection by the temperate phage they released. Some phages infected none of the strains. Phages of Oenoc. oeni had a classical morphology, an isometric head, and a long striated tail. With the broadest host strain as an indicator, phages were detected in wines after malolactic fermentation. Received: 28 November 1997 / Accepted: 5 January 1998     

Immobilization of bacteriophages on gold surfaces for the specific capture of pathogens

Techniques for the chemical attachment of wild-type bacteriophages onto gold surfaces and the subsequent capture of their host bacteria have been developed. The surfaces were modified with sugars (dextrose and sucrose) as well as amino acids (histidine and cysteine) to facilitate such attachment. Non-specific attachment was prevented by using bovine serum albumin as blocking layer. Surfaces modified with cysteine (and cysteamine) followed by activation using 2% gluteraldehyde resulted in an attachment density of 18 ± 0.15 phages/μm². This represented a 37-fold improvement compared to simply applying physisorption. Subsequently, the phage immobilized surfaces were exposed to the host E. coli EC12 bacteria and capture was confirmed by fluorescence microscopy. We obtained a bacterial capture density of 11.9 ± 0.2/100 μm², a 9-fold improvement when compared to those on physically adsorbed phages. The specificity of recognition was confirmed by exposing similar surfaces to three strains of non-host bacteria. These negative control experiments do not show any bacterial capture. In addition, no capture of the host was observed in the absence of the phages.     

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life‐threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 10⁸ pfu ml^?1 of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (10⁶ and 10² cfu ml^?1). In contrast, broth inoculated with 10⁴ phage and 10² bacteria per ml first showed normal bacterial growth until reaching a cell titre of 10⁵ cfu ml^?1. Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml^?1. Phages could be produced with titres of 10¹⁰ pfu ml^?1 in broth culture, but they were not stable upon freeze‐drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.     

The inhibition of bactericidal activity of sera of newborn germ-free piglets to roughEscherichia coli by yeast mannan

The yeast phosphomannan (PM) derived fromHansenula capsulata strain exerts an inhibitory effect on thein vitro bactericidal activity of fresh sera of newborn, colostrum-deprived germ-free piglets to rough strains ofEscherichia coli (S-16 and Lilly). The experiments presented indicate that the PM function probably takes place at the C1 level. The inhibitory effect of PM does not occur provided bacteria are sensitized by specific antiserum prior to exposure to piglet serum. The antibody which was responsible for removal of PM blockade was of 19S nature, 2-mercaptoethanol-sensitive and can be absorbed by heat inactivated bacteria (roughEscherichia coli) or inhibited by addition of soluble somatic antigen (endotoxin) obtained from the same strain ofEscherichia coli (rough). The possible mechanism of inhibition of bactericidal activity by PM is discussed. This investigation was done in the Laboratory of Dr. M. A. Leon, Pathology Research, St. Luke's Hospital, Cleveland, Ohio, U.S.A.     

Recombinant antigens for immunodiagnosis of cystic echinococcosis

Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE) and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The three representative HM-phages (HM 2, HM 3 and HM 7) of Clostridium saccharoperbutylacetonicum were used.

The adsorption rate of the phages HM 2 and HM 7 on the host bacteria was high, whereas that of the phage HM 3 was lower. The adsorption rates of the phages were maximum at pH 5.9~6.6, 30°C.

One-step growth experiment was successfully adapted to the phage-host systems of anaerobic bacteria by bubbling pure nitrogen gas into the medium in the growth tube. The growth characteristics of the HM-phages were investigated by using this technique. The minimal latent periods for phages HM 2, HM 3 and HM 7 were about 45, 90 and 120 minutes, respectively. The corresponding average burst sizes were approximately 500, 100 and 20, respectively. The growth of the phages was optimal at pH 6.2, 30~33°C. The phages failed to grow at 37°C, although the host bacteria multiplied at that temperature. By using a defined medium, it was found that calcium ion was not essential for the growth of the HM-phages.     

Studies on Temperate Phages of Lactobacillus salivarius

Twenty-three temperate phages of Lactobacillus salivarius isolated from human feces were studied as to their morphological, biological, and serological properties. (1) Among 30 strains of L. salivarius tested, 23 strains were lysed by induction with mitomycin C (MC). In all these lysates, phage particles were detected by electron microscopic examination. (2) These phages were morphologically divided into three groups: particles with a regular hexagonal head and a long flexible tail; particles having a regular hexagonal head with or without a short tail-like structure; particles with an elongated head and a long noncontractile tail. (3) Only two, phage 223 having an elongated head and phage 227 with a regular hexagonal head and a long noncontractile tail, produced tiny and very turbid plaques on several host bacteria. Six phages could produce only inhibition zones, ranging from complete inhibition through partial inhibition to normal growth by a serial dilution spot test. (4) All these killer particles could also inhibit the growth of their producer cells. (5) A serological relationship was observed between temperate phages and killer particles, and this was somewhat consistent with the morphological groupings.     

Enumeration of Bacteriophages and Host Bacteria in Sewage and the Activated-Sludge Treatment Process

Bacteriophage populations in an activated-sludge sewage treatment plant were enumerated. A newly developed assay for quantitation of total phages, employing direct electron microscopic counts, was used in conjunction with the plaque assay. The total concentration of phages was significantly higher in reactor mixed liquor and effluent than in influent sewage, indicating a net production of phages within the reactor. Maximum total phage concentrations in the fluid phase of sewage, activated-sludge mixed liquor, and reactor effluent were 2.2 × 10⁷, 9.5 × 10⁷, and 8.4 × 10⁷/ml, respectively. Conditions were optimized for isolation of predominant heterotrophic aerobic bacteria from sewage and mixed liquor. Blending at ice water temperatures was superior to ultrasound or enzyme treatments for maximum release of viable bacteria from microbial floc. A solidified extract of mixed liquor was superior to standard media for cultivating maximum numbers of heterotrophic bacteria. The highest culture counts for sewage and mixed liquor were 1.4 × 10⁷ and 1.3 × 10⁹/ml, respectively, which represented only 3 and 6.8% of the total microscopic cell counts. Only 3 out of 48 dominant bacterial isolates from either mixed liquor or sewage were hosts for phages present in the system. The sum of phage populations infecting these three hosts accounted for, at best, 3.8% (sewage) and 0.2% (mixed liquor) of the total number of phages present. Generally, specific phage titers were lower in mixed liquor than in sewage, indicating that these hosts were not responsible for the net production of phages in the reactor. This study emphasizes the limitations of the plaque assay for ecological studies of phages, and it suggests that bacteria responsible for phage production in activated-sludge mixed liquor are either minor components of the heterotrophic population, floc-producing strains, or members of other physiological groups.     

血清PCT、CRP及内毒素在细菌性血流感染所致脓毒症患者中的早期诊断价值

目的:探究血清降钙素原(PCT)、C反应蛋白(CRP)及内毒素在革兰阳性(G+)杆菌与革兰阴性(G-)球菌血流感染所致脓毒症患者中的早期诊断价值。方法:回顾性分析2010年5月~2015年5月期间我院收治确诊的细菌性血流感染所致脓毒症患者123例,测定其血清PCT、CRP及内毒素水平,通过受试者工作特征曲线(ROC)曲线探究三者对细菌性血流感染所致脓毒症的评估价值。结果:血样培养结果显示,35例患者感染G+菌,88例患者感染G-菌;G-菌组患者血清PCT、CRP及内毒素水平均显著高于G+菌组(P0.05);且G+菌组、G-菌组及所有细菌组患者血清PCT、CRP、内毒素间均呈正相关关系(P0.05);ROC曲线显示,血清PCT、CRP和内毒素诊断G+菌血流感染所致脓毒症患者的截断值分别为1.58μg/L、95.25 mg/L与16.71ng/L,其灵敏度和特异度别为(65.92%,88.37%)、(67.39%,84.38%)与(56.34%,78.93%),诊断G-菌血流感染所致脓毒症患者的截断值分别为2.45μg/L、79.45 mg/L与15.54 ng/L,其灵敏度和特异度别为(78.73%,97.13%)、(68.89%,92.38%)与(65.39%,95.33%)。结论:检测血清PCT、CRP、内毒素水平有利于鉴别G-菌和G+菌血流感染所致脓毒症患者,且敏感度、特异度均较高,可用于早期诊断细菌性血流感染所致脓毒症。